Harnessing diverse transcriptional regulators for natural product discovery in fungi

Abstract

Covering: Up to March 2019

Secondary metabolites (SMs) are chemical entities produced by organisms in response to environmental stimuli and as a defense against biological warfare. The production of SMs is controlled by a hierarchical regulatory network involving core factors that orchestrate transcriptional activation of SM gene clusters. In the past few years, significant achievements have been made in the discovery of novel fungal natural products by genetic manipulations of various types of transcriptional regulators. In this review, we summarized the representative regulators for the activation of fungal secondary metabolism and focused on the strategies for the exploitation of these regulators and their application in finding novel structures.

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Hai-Ning Lyu

Hai-Ning Lyu obtained his Bachelor's degree (2011) in Pharmaceutical Engineering from Beijing University of Chinese Medicine. He received his Master's (2014) and PhD degree (2017) in Natural Medicine at Peking University, under the supervision of Prof. Peng-Fei Tu and Prof. Yong Jiang. His doctoral research focused on the discovery of bioactive natural products from plants of the genera Murraya and Sinomenium. Currently, he works as a staff scientist in the Institute of Microbiology, Chinese Academy of Sciences, with Professor Wen-Bing Yin, where his research interests include the discovery, biosynthesis and metabolic engineering of natural products.

Hong-Wei Liu

Hong-Wei Liu is currently a Professor in the Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing, China. He obtained his PhD in medicinal chemistry in 2003 from Shenyang Pharmaceutical University with Professor Xin-Sheng Yao where he investigated the bioactive natural products from traditional Chinese medicine. Then he worked with Professor Guo-Ping Cai as a post-doctoral fellow at the Tsinghua University to study marine natural products. After that, he moved to Capital Medical University where he worked as an associated professor in the School of Pharmaceutical Sciences. In 2009, he joined the Institute of Microbiology and led a research team in the field of fungal natural products. His research interests mostly focus on the identification of novel bioactive fungal secondary metabolites, elucidation of biosynthetic pathway of fungal products, and generation of bioactive pharmaceutical agents by synthetic biology methods.

Nancy P. Keller

Nancy Keller obtained her PhD degree in the Plant Pathology Department at Cornell University and is currently a Professor in the Department of Medical Microbiology and Immunology and Bacteriology at the University of Wisconsin. For her entire professional career, she has explored the genetics underlying fungal development with an emphasis on natural product synthesis in fungi.

Wen-Bing Yin

Wen-Bing Yin is currently a Professor in the Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing, China. He obtained his PhD in Pharmaceutical Biology in 2009 from Philipps-University Marburg with Professor Shu-Ming Li where he studied the chemo-enzymatic biosynthesis of fungal natural products. Then he worked with Professor Nancy Keller as a post-doctoral fellow at the University of Wisconsin–Madison to study gene regulation on fungal secondary metabolism. After that, he moved to Los Angeles and worked with Professor Yi Tang at the University of California where he studied the discovery of natural products by a synthetic biological approach. In 2014, he returned to China to join the Institute of Microbiology and was supported by “the 100 Talents Project” of CAS. His research interests mostly focus on the activation, biosynthesis of silent fungal biosynthetic gene cluster and synthetic biological reconstruction of pharmaceutical molecules.

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1. Introduction

Secondary metabolites (SMs), usually called natural products (NPs), play a highly significant role in the discovery of drug leads for the treatment of human diseases. For example, more than half of the clinically approved medicines are NPs or their derivatives.¹ Despite the significant advances made via the study of natural resources, industrial investments in NP research have been reduced as traditional NP discovery methods (growth of microbes on different media) cannot meet the ever-growing demands for new bioactive molecules in drug research and development; therefore, the development of innovative approaches for accurate targeting and activation of undiscovered NPs is urgent for drug discovery.

Filamentous fungi are well known for their abilities to produce an enormous amount of SMs, many of which, including antibiotics, cholesterol-lowering drugs, immunosuppressants, and anti-osteoporosis agents, have benefitted human health.^2,3 Recent advances made in sequencing technologies and bioinformatics analysis have revealed a large amount of fungal SM encoding genes, which are usually clustered in a chromosome and called biosynthetic gene clusters (BGCs).^4–6 However, to date, only a few BGCs have been functionally annotated and correctly assigned to the relative metabolites;^4–9 one of the major reasons hindering the identification of BGCs is the poor understanding of how these BGCs are regulated. Most BGCs are not expressed or only lowly expressed under laboratory culture conditions; this suggests that a huge fungal NP treasure trove is yet to be exploited. Indeed, it has been estimated that several million fungal BGCs are yet to be characterized.¹⁰

How to activate these BGCs in the laboratory? In recent years, genome mining strategies, including transcriptional regulator modulation, promoter engineering, as well as heterologous expression, have been successfully developed and introduced to awake the silent/lowly expressed BGCs for new fungal NP discovery.^10,11 Among them, genetic manipulations of regulators have made significant progress, ranging from overexpression of pathway-specific regulators to deletion of epigenetic blocks of BGC expression to gain access to new fungal NPs. In this review, we presented the recent achievements made in mining novel NPs in fungi via genetic manipulations of various types of regulators. The inherent advantages and disadvantages of these approaches have also been discussed. Moreover, a promising combinational strategy for the discovery of novel fungal SMs has been outlined in Section 4.

2. Transcriptional regulators play critical roles in fungal secondary metabolism

Fungi produce SMs to adapt to variable environmental stresses, and thus, the production of SMs is controlled by complex regulatory networks. The current understanding of fungal secondary metabolism regulation has been reported in many excellent reviews,^{7–9,11–14} indicating that regulators are central players in the fungal secondary metabolism. In this section, we present the critical roles of regulators involved in SM biosynthesis by outlining the fungal SM regulatory network at different levels including pathway-specific regulation, global regulation, and epigenetic regulation.

More than half of the fungal BGCs contain a putative regulatory gene.⁸ These transcription factors act as pathway-specific regulators that connect the fungal BGCs to one specific metabolite (as shown in Scheme 1). The most common type of these regulators is the Zn(II)₂Cys₆ domain protein that has to date only been found in fungi. The archetype is undoubtedly AflR, which binds to the palindromic sequence 5′-TCG(N5)CGA-3′ in the promoter regions of several aflatoxin and sterigmatocystin cluster genes in Aspergillus.¹⁵ In addition to Zn(II)₂Cys₆ proteins, Cys₂His₂ zinc-finger proteins are commonly found in fungi such as Tri6 for the regulation of mycotoxin deoxynivalenol (DON) with Tri10 by cAMP signalling in F. graminearum.¹⁶ The less frequently found regulators in fungi are ankyrin repeat proteins (ToxE for HC-toxin production)¹⁷ and winged helix proteins (CPCR1 for cephalosporin C production).¹⁸ However, all these transcription factors were first identified in laboratory-expressed BGCs; moreover, they were also present in laboratory-silent BGCs and successfully used to activate new metabolites, as has been described in the next section.

Scheme 1 The strategy for fungal NP discovery via genetic manipulation of regulators. (A) Shows the possible locations of three types of regulators (pathway-specific regulator, global regulator, and epigenetic regulator) within the fungal genome, marked in red. (B) Shows the generation of SMs 1 and 3 under standard culture condition for demonstration. (C) Displays four hypothetical SM profiles of mutants. Manipulations of pathway-specific regulators led to the production of the corresponding SMs 2 and 4. Manipulations of global and epigenetic regulators could change the whole SM metabolic profiling, which activated the silent SMs 2 and 4 randomly.

Pathway-specific regulation delineates the transcription of single BGCs in quite a direct pattern. However, fungal SM biosynthesis is generally affected by various environmental factors including pH, temperature, nutrition supply, interspecific interactions, and other unknown factors. Global regulators often relay these environmental signals through higher-level regulation of the fungal secondary metabolism (as shown in Scheme 1). For example, aflatoxin and sterigmatocystin production is decreased with the increasing environmental pH.¹⁹ The overexpression of pacC, a global regulator for pH regulation in fungi, could mimic alkaline conditions for reducing the productions of aflatoxin and sterigmatocystin in A. nidulans.¹⁹ PacC acts as a key transcription mediator in the pH regulation of various BGCs via repressing the transcription of acid-expressed genes. To date, a number of global regulators, such as CreA (carbon catabolite regulation and regulation of patulin biosynthesis in Penicillium expansum),^20,21 AreA (nitrogen regulation and regulation of up to 1/2 of the BGCs in Fusarium fujikuroi),^22,23 and the HAP-like CCAAT-binding complex regulating penicillin production and iron supply,^24,25 responding to environmental signals and involved in the regulations of specific BGCs have been identified. Genetic manipulation of these regulators influences the production of SMs that are susceptible to the corresponding environmental stimuli. However, note that global regulators usually participate in the huge and complex regulatory network of fungal development; thus, genetic operations on these types of regulators may cause abnormal growth and/or dramatic changes in the metabolomic profile of fungi.

Although the co-regulation of SM biosynthesis by pathway-specific and global regulators depicts a simple multiple level model system at first glance, fungal SM regulation does not follow a strict hierarchical regime in most cases. For example, the discovery of LaeA—a global regulator positively regulating up to half of the BGCs in filamentous fungi^26–29—has led to the finding that SMs are intimately connected to fungal development. A seminal study reported in 2008 demonstrated that LaeA partnered with two other proteins to form the ‘Velvet Complex’ composed of VelB, VeA, and LaeA, coordinating fungal development and secondary metabolism in response to light.³⁰ Unlike the other global regulators that are bonafide transcription factors, LaeA is a nuclear protein with the closest identity to arginine and histone methyltransferases; this suggests that LaeA may affect the SM production via epigenetic modification of the chromatin. This hypothesis was further supported by comparison of the heterochromatic marks of the sterigmatocystin BGC in the wild-type (high sterigmatocystin) versus the ΔlaeA mutant (low sterigmatocystin) in A. nidulans; this revealed increased repressive histone 3 lysine 9 (H3K9) trimethylation in the ΔlaeA mutant.³¹ Therefore, the discovery of LaeA demonstrated a chromatin-mediated mechanism for secondary metabolism regulation and opened a new strategy by epigenetic manipulation of chromatin modifying proteins to genetically control the expressions of fungal BGCs.

3. Regulators as tools to shed light on the new fungal chemistry

Among the regulators abovementioned in Section 2, the most useful tools for fungal NP discovery are pathway-specific regulators and chromatin modifying proteins. To date, a number of silent fungal SMs, including both truly new compounds and known metabolites (which have been isolated from other fungi before), have been obtained upon BGC activation. The structures of the truly new compounds are shown in Fig. 1, and their regulators-in-charge are listed in Table 1.

Fig. 1 Novel fungal NPs produced by genetic manipulation of pathway-specific (1–30), global (31–37), and epigenetic (38–79) regulators. *: BGC not identified.

Table 1 Regulators used for discovering new fungal NPs

Regulators^a	Accession no.	Domain/description	New metabolites discovered	Species	References
a Alphabetically arranged. b Underdetermined. c BGC not identified.
Pathway-specific regulators
AfoA	Q5BEK1	Zn(II)2Cys6	Asperfuranone 3	Aspergillus nidulans	33
AoiH	BAE65966	Zn(II)2Cys6	12	Aspergillus oryzae	37
ApdR	Q5ATG6	Zn(II)2Cys6	Aspyridones A 1 and B 2	Aspergillus nidulans	32
APS2	S0EBQ6	Zn(II)2Cys6	Apicidin F 19	Fusarium fujikuroi	41
AzaR	G3XMC5	Zn(II)2Cys6	Azanigerones A–F 4–9	Aspergillus niger	34
BerA	—^b	Zn(II)2Cys6	Berkeleyacetal D 14, 11-epiberkeleyacetal C 15	Neosartorya glabra	39
CaaR	CAK45414	Zn(II)2Cys6	Carlosic acid methyl ether 11	Aspergillus niger	36
FSL7	ESU17175	Zn(II)2Cys6	Fusarielins F–H 16–18	Fusarium graminearum	40
NscR	A1D8J1	Zn(II)2Cys6	Neosartoricin 13	Aspergillus fumigatus	38
PynR	CAK48262	Zn(II)2Cys6	Pyranonigrin E 10	Aspergillus niger	35
FsqA	XP_747734	Zn(II)2Cys6	Fumisoquins A–C 20–22	Aspergillus fumigatus	42
XanC	A4DA05	bZIP	Melanocins E 23 and F 24	Aspergillus fumigatus	43
—	EAA66632	Zn(II)2Cys6	25	Aspergillus nidulans	44
—	EAA64857	Zn(II)2Cys6	26	Aspergillus nidulans	44
—	EAA63353	Zn(II)2Cys6	27–30	Aspergillus nidulans	44
Global regulators
LaeA	EAQ93455	—	Chaetoglobosin Z 31	Chaetomium globosum	55
LaeB	EAA60741	—	3-Methoxyporriolide 32, 7-methoxyporriolide 33, gibellulin C 34^c and D 35^c	Aspergillus nidulans	56
McrA	EAA60243	Zn(II)2Cys6	36, 37	Aspergillus nidulans	52
Epigenetic regulators
Hat1	EFZ02700	Histone acetyltransferase	Meromusides A–I 63–71^c, meromutides A 72^c and B 73^c	Metarhizium robertsii	72
HdaA	—	Histone deacetylase	Arbumycin 42, arbumelin 43, arbusculic acids A 44 and B 45	Calcarisporium arbuscular	70
NnaB	CBF84101	GNAT-type acetyltransferase	Pheofungins A–D 38–41	Aspergillus nidulans	61
PfcclA	ETS83251	Histone methyltransferase	Pestaloficiols T–V 59–61^c	Pestalotiopsis fici	71
PfhdaA	ETS79135	Histone deacetylase	Ficiolides A–K 48–58^c, pestaloficiol W 62^c	Pestalotiopsis fici	71
RpdA	EAA60836	Histone deacetylase	Aspercryptins A1 46 and A2 47	Aspergillus nidulans	65
Other regulators
PfcsnE	ETS75687	COP9 signalosome	Pestaloficins A–E 74–78^c	Pestalotiopsis fici	75
PkaC1	CAC82611	Protein kinase A	Fumipyrrole 79	Aspergillus fumigatus	76

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3.1 Pathway-specific transcription factors activate the dedicated BGCs

Manipulation of the pathway-specific regulators is one of the most convenient and successful approaches to discover novel NPs. These transcription factors are encoded within the BGC they regulate and thus easily recognized bioinformatically. Several cryptic BGCs have been activated, and new metabolites have been identified simply by overexpressing the BGC regulator. For example, the novel pyridone alkaloids aspyridones A and B (1 and 2) were identified when the in-cluster putative pathway-specific regulator apdR (a Zn(II)₂Cys₆ protein) was overexpressed and activated the entire apd BGC in A. nidulans (Fig. 1).³² In the same fungus, the overexpression of the transcriptional regulatory gene afoA in a BGC including two PKS genes resulted in the production of a new polyketide, asperfuranone 3 (Fig. 1).³³ In A. niger, the overexpression of azaR, a Zn(II)₂Cys₆ zinc finger protein within a dual PKS gene cluster, afforded six novel azaphilone compounds, azanigerones A–F (4–9) (Fig. 1).³⁴ Similarly, by applying the same method, two PKS/NRPS hybrid metabolites (pyranonigrin E 10 and carlosic acid methyl ether 11) (Fig. 1) were characterized in the same species.^35,36 To date, ca. 30 new compounds have been discovered via overexpression of pathway-specific regulators. These include polyketide 12 from A. oryzae,³⁷ anthracenone 13 from A. fumigatus,³⁸ the berkeleyacetal derivatives 14 and 15 from Neosartorya glabra,³⁹ cytotoxic fusarielins 16–18 from F. graminearum,⁴⁰ apicidin congener 19 from F. fujikuroi,⁴¹ fumisoquins A–C 20–22 from A. fumigatus,⁴² the melanocins E 23 and F 24 from A. fumigatus,⁴³ and the polyketides 25–30 from A. nidulans⁴⁴ (Fig. 1).

Indeed, the manipulation of pathway-specific regulators is a straightforward method to activate a specific BGC in fungi and offers unique advantages by accurately targeting novel structures even from chemically well-studied species. However, this approach has limitations if a BGC doesn't possess a pathway-specific regulator or does not activate for some unknown reason. In these cases, combinational methods with other genetic manipulation techniques can be used to activate a BGC. For example, replacement of the promoters of BGC genes led to the identifications of eight polyketide compounds, including the novel structures 25–30 in A. nidulans.⁴⁴ An exciting advance was achieved with the activation of the asperlin BGC in A. nidulans through fusion of the DNA-binding domain of the silent asperlin cluster with the asperfuranone transcription factor AfoA.⁴⁵

3.2 Global regulators can enhance the overall fungal NP diversity

Genetic mutagenesis has been successful in identifying several global regulators of fungal secondary metabolism. For instance, laeA and laeB were identified from A. nidulans by examining 23 mutants that displayed a phenotype of loss of sterigmatocystin production.^26,46McrA was disclosed through screening the mutants that up-regulated the transcription of the BGC for nidulanin A biosynthesis. In general, the global regulators are conserved across fungal genera. By identifying the sequence of each protein, such as LaeA,^26,47 LaeB,⁴⁶ CreA,²⁰ AreA,²² PacC,⁴⁸ MeaB,⁴⁹ HapX,⁵⁰ RsmA,⁵¹ and McrA,⁵² we can find a homolog in most ascomycetes and thus use that gene to up-regulate the BGCs in the selected fungi. In particular, LaeA has been identified and manipulated in dozens of fungal species; this has led to the studies of a magnitude of BGCs.⁵³

Basically, the manipulation of global regulators for new NP discovery could be regarded as a “genetic” form of the OSMAC⁵⁴ (One Strain Many Compounds) approach. Note that the studies concerning global regulators mainly focus on their influences on fungal development or the production changes of known SMs such as the stimulation of antibiotic yield,^7,8 rather than the discovery of new NPs. However, some studies have showed new metabolites using these proteins. For example, the overexpression of laeA up-regulated the expression of the chaetoglobosin gene cluster in Chaetomium globosum and led to the isolation of a new structure, chaetoglobosin Z 31, together with six known analogues, chaetoglobosins A, B, D, E, O, and V.⁵⁵ Recently, eight silent SMs have been activated and obtained from A. nidulans by disruption of LaeB.⁵⁶ Among the isolates, two phthalides (3-methoxyporriolide 32 and 7-methoxyporriolide 33) and two dibenzo[1,4]dioxins (gibellulin C 34 and D 35) possessed new structures. This was the first study on dibenzo[1,4]dioxin derivatives obtained from an Aspergillus species, enhancing the chemical diversity of the genus. Deletion of the global regulator mcrA in A. nidulans led to the dramatic upregulation of nidulanin derivatives and prenyl xanthones, along with two new cichorine analogues 36 and 37 (Fig. 1).⁵²

3.3 Epigenetic regulators in the fungal natural product discovery

Similar to the case of global regulators, the genetic manipulation of epigenetic regulators can activate a multitude of BGCs that are often laboratory-silent BGCs. The reader is referred to a recent review for extensive details on this approach.⁵⁷ The success of this method is due to chromatin remodelling arising from the deletion or overexpression of histone modifying genes that lead to the activation of the BGCs located in the heterochromatic regions on the chromatin. For example, Bre2 is a critical member of the COMPASS complex, which is a conserved eukaryotic transcriptional effector bidirectionally regulating chromatin-mediated processes through methylation of H3K4.⁵⁸ Deletion of cclA in A. nidulans, the ortholog of bre2, activates the production of emodin analogues as well as two polyketides F9775A and B, whose encoding BGCs are located at the right telomere of chromosome VIII and the left telomere of chromosome II, respectively.²

By knowing that histone proteins function as a scaffold for nucleosome formation, modifications of histone decorations, including methylation, acetylation, phosphorylation, ubiquitylation, and sumoylation,⁵⁹ have been proven to be dependable approaches for remodelling of the chromatin landscape and, in the case of fungi, activating BGCs.⁵⁷ The most common strategy is to delete or overexpress histone methyltransferases, acetylases, demethylases or deacetylases including GcnE,⁶⁰ NnaB,⁶¹ EsaA,⁶² HdaA,⁶³ SirA,⁶⁴ RpdA,⁶⁵ HosA,⁶⁶ HstD,⁶⁷ KdmA,⁶⁸ and RmtA.⁶⁹ Some of these manipulations have been successfully employed to access fungal chemical diversity and new NP discovery. Deletion of the putative GNAT-type acetyltransferase NnaB in A. nidulans resulted in a slow-growing, yellow to red/orange pigmented strain that resulted in a significantly increased production of SMs. HPLC-HRESIMS analysis of the ΔnnaB strain yielded a series of heterocyclic products, which were further identified as pheofungins A–D (38–41) (Fig. 1).⁶¹ Inactivation of the histone deacetylase hdaA resulted in aberrant growth and physiology in Calcarisporium arbuscular as well as production of novel metabolites, two new peptides (arbumycin 42 and arbumelin 43) and two new diterpenes (arbusculic acids A 44 and B 45) (Fig. 1).⁷⁰ Similarly, down regulation of the histone deacetylase rpdA in A. nidulans led to the selective up-regulation of two new lipopeptides, aspercryptin A1 46 and A2 47 (Fig. 1).⁶⁵ Chemical analysis of the HdaA and CclA deletion mutants in P. fici revealed polyketide productions of 11 macrodiolide ficiolides A–K (48–58) as well as pestaloficiols T–W (59–62) (Fig. 1).⁷¹ Ficiolide K 58 was found to contain a very rare 1,6-anhydropyranose moiety. Moreover, deletion of the putative histone acetyltransferase Hat1 in Metarhizium robertsii resulted in the characterizations of 11 new polyketides, meromusides A–I (63–71) and meromutides A 72 and B 73 (Fig. 1).⁷²

All the abovementioned studies involved the genetic manipulation of the genes encoding enzymes that add or remove chemical moieties on histone tails. Most recently, another class of histone modifying proteins, the histone reader SntB, has been found to modify the BGC expression.⁴⁶ By deleting sntB in A. flavus, seven known NPs and many uncharacterized secondary metabolites were aberrantly regulated.⁷³ Moreover, in addition to genetically modifying epigenetic regulators, treatment with chemicals, such as DNA methyltransferase and histone deacetylase inhibitors, can control the native expression of fungal biosynthetic pathways and generate new bioactive SMs. Readers that are interested in chemical epigenetic methodologies for exploring novel NPs are referred to the reported literature.⁷⁴

3.4 Other regulators as tools in accessing the structural diversity

In addition to the abovementioned regulators, several alternative regulator-based approaches can be used to discover new NPs. The COP9 signalosome (CSN) is a highly conserved protein complex implicated in diverse biological functions that involve ubiquitin-mediated proteolysis. By disrupting the fifth subunit of CSN in P. fici, the resultant strain ΔPfcsnE lost the ability to produce conidia along with remarkable changes in the SM output. In addition, five new structures, pestaloficins A–E (74–78), were thus isolated and characterized (Fig. 1).⁷⁵ Pestaloficin A 74 represents a new type of dimeric cyclohexanone derivative linked through an unprecedented pentacyclic spiral ring. Cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) is one of the most studied signal transduction pathways that regulate gene expression and activation of secondary metabolism in fungi. Overexpression of the gene coding for the PKA catalytic subunit (PkaC1) increased the expressions of the fmp cluster genes in A. fumigatus and led to the isolation of a new NP, fumipyrrole 79.⁷⁶

4. Combinational regulatory strategy to elucidate new fungal chemistry

As abovementioned, different regulator classes exhibit unique mechanisms by which new fungal chemistry can be accessed; for example, while the manipulation of both global and epigenetic regulators result in the alteration of the expression of multiple BGCs in a fungus, the mechanisms are quite different: one largely occurs through alteration of transcriptional networks and the other occurs through remodelling of chromatin; targeting of pathway-specific regulators results in the accurate activation of a specific BGC. Moreover, SM production is a complicated process because these metabolites are dedicated to the ecological or physiological functions of the fungus. Thus, fungal NP production is also controlled by various spatial and temporal inputs.¹⁴ Therefore, a universal system that integrates multiple regulators into a single model fungal host would be a powerful route to uncover novel fungal NP treasures. Herein, we proposed a combinational strategy for the effective and accurate discovery of new NPs in fungi.

As shown in Scheme 2, this strategy combines the integration of global/epigenetic regulators and pathway-specific regulators at different levels. As a demonstration, a BGC with no in-cluster transcriptional factor is selected. In Aspergillus spp., the expression of lovastatin is directly controlled by the pathway-specific regulator LovE, whereas the penicillin production is regulated by a higher regulatory network. However, both SMs are dramatically up-regulated in an OE::laeA mutant in Aspergillus spp.²⁶ In this case, the LaeA-lovastatin/penicillin regulation system could be selected as a synthetic DNA gene-expressing UNIT. Using gene switching techniques such as via yeast assembly, the lovastatin and penicillin biosynthetic ORFs would be replaced by target genes of the desired cryptic cluster; this would leave the native promotors in the gene-expressing UNIT. This would allow the upregulation of cryptic genes by LaeA and additionally LovE in the case of using lovastatin promoters. This strategy would enable the BGC expression in the model host regardless of the BGC from any fungal strain. We can anticipate that with the characterization of more key regulators and mechanisms involved in the SM production, more efficient combinational strategies will be developed for mining novel fungal chemical diversity.

Scheme 2 A proposed combinational strategy for activating targeted silent BGCs, using LaeA and LovE as a case study. (A) The expressions of the lovastatin (also containing a pathway specific transcription factor LovE) and penicillin (no specific transcription factor) gene clusters are dramatically up-regulated by overexpression of the global regulator LaeA in Aspergillus spp.²⁶ (B) The hypothetical heterologous universal cluster expression UNIT. The LaeA/LovE regulation system can be utilized to substitute genes from BGCs of interest for penicillin or lovastatin ORFs in a tunable LaeA host strain.

5. Conclusions and prospects

With the advent of cheap sequencing and bioinformatics technologies, enabling the identification of a huge number of BGCs in fungal genomes, there is now an unprecedented potential for discovering specific SMs in fungi.^4,5,10 Among the diverse genome-based strategies for exploring novel NPs, regulator manipulation exhibits a unique advantage: only one gene needs to be handled, avoiding the complicated genetic manipulation required for whole BGC pathway reconstruction. In this review, we report a total of 79 completely new compounds, including the biologically interesting compounds aspyridones A and B (1 and 2),³² the bioactive molecules fusarielins (16–18)⁴⁰ and apicidin F 19,⁴¹ and the new skeleton structure pestaloficin A 74,⁷⁵ discovered by applying these strategies. However, as in the case of any technology, there is still a need for improvement. For example, these achievements have been limited to fungi that are genetically accessible. In this regard, one future goal is to improve the efficiency of genetic manipulations in all fungi; possibly with the advent of the CRISPR technology, there is foreseeable improvement in this area.⁷⁷ Moreover, a better understanding of the regulatory networks controlled by global or epigenetic regulators would result in new methods to harness this network to accurately activate silent NPs through alternative means. Keeping these challenging goals in mind, it is clear that regulator manipulation will continue to be a powerful and efficient tool for new NP discovery in fungi.

6. Conflicts of interest

We declare no conflicts of interest.

7. Acknowledgements

We thank Professor Shu-Ming Li (Philipps University of Marburg) for critical reading of the manuscript. This project was financially supported by the National Key Research and Development Program (2016YFD0400105), National Natural Science Foundation of China (31861133004), the Construction of the Registry and Database of Bioparts for Synthetic Biology of the Chinese Academy of Sciences (ZSYS-016) and the State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (Grant GTZK201802). Wen-Bing Yin is a scholar of “the 100 Talents Project” of CAS.

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